*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

It definitely made my day to read, "Long after it should have been apparent, we realized that this aberration is a result of tilt of the coverslip."  This is such a classic moment in science, to the point that every paper, if the authors were being brutally honest, would have a sentence to the effect of, "Long after it should have been apparent, we realized that...[insert discovery that seems obvious in hindsight, but no one initially anticipated]."

Carry on,
   Ben Smith

Benjamin E. Smith, Ph.D.
Samuel Roberts Noble Microscopy Laboratory
Research Scientist, Confocal Facility Manager
University of Oklahoma
Norman, OK 73019
E-mail: [log in to unmask]
Voice   405-325-4391
FAX  405-325-7619
http://www.microscopy.ou.edu/

________________________________________
From: Confocal Microscopy List [[log in to unmask]] on behalf of John Oreopoulos [[log in to unmask]]
Sent: Tuesday, April 21, 2015 7:18 AM
To: [log in to unmask]
Subject: Re: PSF asymetry

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Just a guess here, maybe the nosepiece is not engaging properly (ie: objective is no co-linear with the optic axis). A mis-aligned tube lens? What happens if you take the objective(s) to another microscope?
What about the microscope stage? Could it be grossly tilted in some manner? Some stages come with an adjustable stage insert to make the sample coverslip perpendicular to the optic axis.

You mentioned it was a TIRF objective, so I imagine you use this with immersion oil and a sample embedded in water. However, your images do look reminiscent of the abberation mentioned in this paper:

Arimoto, R. and J.M. Murray, A common aberration with water-immersion objective lenses. Journal of Microscopy-Oxford, 2004. 216: p. 49-51.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca


On 2015-04-21, at 4:10 AM, Christophe Leterrier wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Dear microscopists,
>
> I am using a very nice TIRF microscope with a 100X, 1.49 NA objective. I
> had the impression that there was a slight lateral shift when defocusing up
> and down, so I checked the PSF with 100 nm beads on a HR #1.5 coverslip.
> What appears on the attached image (three planes taken at -1, 0 and +1 um)
> is that the PSF is not rotationnally symetric, i.e.more intense on the
> left-bottom side :
>
> https://drive.google.com/file/d/0B_JeGjE7nBHWWFViM0pwSy0xR3M/view
>
> This asymetry is quite constant over the field of view  (it is not radial
> relative to the center of the field). It does not depend on the
> illumination (it is the same under azimutal laser, TIRF laser,
> epifluorescence lamp). It does not depend on the filter cube used. Finally
> (and this is what surprises me the most), I got another brand new 100X,
> 1.49 objective for testing and it still shows up (the attached image is
> taken with the new objective).
>
> Do you have an idea if what could be wrong, and how to correct it? Could it
> be caused by an internal lens? By the sample used?
>
> Thanks for your help,
>
> Christophe